Epigenomics studies represent the next wave of efforts to decode the information embedded in genomes beyond the nucleotide sequences. Dynamic epigenomic changes, such as DNA methylation are key processes in cell differentiation, development and many pathological events. Current methods for mapping epigenetic modifications on DNA, such as bisulfite sequencing or using methylation-sensitive restriction enzymes, are either technically challenging or provide incomplete information. The proposed research in this grant application is based on a novel family of modification-dependent restriction endonucleases (REs), represented by MspJI, which we have discovered recently. Unlike other existing REs, these enzymes recognize modified cytosines in DNA and cleave at fixed distances away from the recognition sites. One of their unique properties is that they are capable of releasing short DNA fragments containing the methylated cytosines directly from the genomic DNA. Using ultra high- throughput sequencing platforms, one would be able to identify and map epigenetic modification in a reliable and quick manner. Furthermore, a significant portion of the modified cytosines in mammalian cells is in the form of 5-hydroxymethylcytosine and current methods cannot determine their presence. MspJI in combination with a 5- hydroxymethylcytosine modifying enzyme, such as beta-glucosyltransferase, can distinguish such changes. Thus, application of these enzymes can provide a foundation for the next-generation of methods for analyzing epigenomic modification. In the Phase I research, we plan to purify the recombinant enzymes and characterize their biochemical properties in detail in vitro. In the Phase II research, we plan to determine the molecular structure of the enzymes both in their apo-forms without DNA and complexed with a methylated DNA substrate. We will establish methodologies whereby these enzymes can be used to decode the DNA methylation patterns in human, mouse and a few other model organisms. We will also examine the dynamics of DNA methylation during mouse embryonic stem cell differentiation. The availability of these enzymes will allow us to answer scientifically pressing questions, such as, the sequence location of 5-hydroxymethycytosine in the human and mouse genomes. Another goal of the Phase II research is that, based on the structures and our previously established enzyme engineering protocols, we plan to isolate mutants that may have improved properties. We believe our proposed research lays out a few unique and exciting opportunities for epigenetics research and we expect the availability of these enzymes as products and as parts of kits to have a major impact for the broader biomedical community interested in studying epigenetic modifications. PUBLIC HEALTH RELEVANCE: Epigenetic DNA modifications in mammalian genomes, especially methylation, play crucial roles in gene regulation during cell differentiation. Commonly used methods, such as bisulfite sequencing, have many inherent drawbacks. The proposed research in this project aims at providing a set of novel enzymatic reagents for mapping the epigenetic landscape based on a family of newly discovered modification-dependent restriction endonucleases. We plan to characterize the biochemical properties of these enzymes and develop methods for applying them in epigenetics research. Coupled with high-throughput sequencing technologies, they promise a much simplified pipeline from which myriads of revolutionary methodologies and research can build on.